ABSTRACT This research will address a major knowledge gap in aryl hydrocarbon receptor (AHR) biology: how transcriptional activation of the AHR by the environmental toxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin, TCDD) causes metabolic toxicities including suppressed glucose production, energy failure and hepatic steatosis. We will investigate the role of NAD+ depletion in AHR metabolic toxicities. NAD+ is a small molecule important in cellular redox reactions and is a required substrate for the catalytic activity of PARPs and sirtuins, enzymes that regulate energy metabolism, genome stability and aging. This research will provide new understanding of the basis of AHR metabolic toxicities by probing our finding that TCDD decreases NAD+ levels through the AHR target gene TiPARP (TCDD-inducible poly ADP-ribosylase, PARP7), one of the PARP enzymes which consume NAD+ while ADP-ribosylating proteins. We will exploit here our discoveries that TiPARP contributes to decreased hepatic glucose production by consuming NAD+ and suppressing induction by sirtuin 1 of PEPCK, a rate limiting enzyme in gluconeogenesis. Longer exposure to TCDD also increases hepatic PARP1 levels suggesting that PARP1 may contribute to lowering NAD+ levels. We propose two connected Specific Aims (SA): SA1 will study the consequences of NAD+ depletion on sirtuin activity and mitochondrial bioenergetics, will establish the role of TiPARP and PARP1 in TCDD toxicities in mammalian model and will seek to identify NAD-repletion as a preventive and corrective strategy against TCDD toxicity. SA2 will examine the role of ADP-ribosylation by TiPARP in AHR action. Specifically, these studies will elucidate the consequences of NAD+ depletion for biological and catalytic activities of Sirts1, 3 and 6, using new sirtuin specific assays (SA1a1). A role of NAD+ depletion by TCDD in mitochondrial bioenergetics will be addressed by studies using Seahorse technology to examine oxygen consumption and glycolysis (SA1a2). Chick embryo hepatocytes will be used throughout the grant, and we will confirm major findings in human primary hepatocytes. Liver-specific TiPARP and PARP1 KO mice will be used to assess their roles in the production of TCDD metabolic toxicities in a mammalian model (SA1b). SA1c will determine whether NAD+ repletion with nicotinamide, and other NAD+ repleting agents, can prevent and possibly correct TCDD toxicities. SA2 will examine the role of ADP-ribosylation by TiPARP in AHR metabolic toxicities as we found that PEPCK is ADP-ribosylated by TiPARP. We will examine (SA2a) the effects of ADP-ribosylation of PEPCK by TiPARP on PEPCK stability and activity and will seek to identify other proteins ADP-ribosylated by TiPARP to reveal new ways by which ADP-ribosylation could participate in AHR action (SA2b). We expect this research to establish a significant mechanism in which NAD+ depletion by PARP activity leads to TCDD hepatic metabolic toxicities and to identify NAD+-repletion as an approach to prevent and/or correct AHR metabolic toxicities in vivo.